Cloning of an Endangered Species (Bos gaurus) Using Interspecies Nuclear Transfer (original) (raw)
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Biology of Reproduction, 2007
In nondomestic and endangered species, the use of domestic animal oocytes as recipients for exotic donor nuclei causes the normal pattern of cytoplasmic inheritance to be disrupted, resulting in the production of nuclear-cytoplasmic hybrids. Evidence suggests that conflict between nuclear and cytoplasmic control elements leads to a disruption of normal cellular processes, including metabolic function and cell division. This study investigated the effects of nuclear-cytoplasmic interactions on the developmental potential of interspecies embryos produced by in vitro fertilization and somatic cell nuclear transfer: cattle 3 cattle, gaur 3 cattle, hybrid 3 cattle. Cattle control and hybrid embryos were examined for development to the blastocyst stage and blastocyst quality, as determined by cell number and allocation, apoptosis incidence, and expression patterns of mitochondria-related genes. These analyses demonstrated that a 100% gaur nucleus within a domestic cattle cytoplasmic environment was not properly capable of directing embryo development in the later preimplantation stages. Poor blastocyst development accompanied by developmental delay, decreased cell numbers, and aberrant apoptotic and related gene expression profiles, all signs of disrupted cellular processes associated with mitochondrial function, were observed. Developmental potential was improved when at least a portion of the nuclear genome corresponded to the inherited cytoplasm, indicating that recognition of cytoplasmic components by the nucleus is crucial for proper cellular function and embryo development. A better understanding of the influence of the cytoplasmic environment on embryonic processes is necessary before interspecies somatic cell nuclear transfer can be considered a viable alternative for endangered species conservation.
Reproduction Fertility and Development, 2007
Somatic cell nuclear transfer (SCNT) can provide a unique alternative for the preservation of valuable individuals, breeds and species. However, with the exception of a handful of domestic animal species, successful production of healthy cloned offspring has been challenging. Progress in species that have little commercial or research interest, including many companion animal, non-domestic and endangered species (CANDES), has lagged behind. In this review, we discuss the current and future status of SCNT in CANDES and the problems that must be overcome to improve preand post-implantation embryo survival in order for this technology to be considered a viable tool for assisted reproduction in these species.
Wildlife conservation and reproductive cloning
Reproduction, 2004
Reproductive cloning, or the production of offspring by nuclear transfer, is often regarded as having potential for conserving endangered species of wildlife. Currently, however, low success rates for reproductive cloning limit the practical application of this technique to experimental use and proof of principle investigations. In this review, we consider how cloning may contribute to wildlife conservation strategies. The cloning of endangered mammals presents practical problems, many of which stem from the paucity of knowledge about their basic reproductive biology. However, situations may arise where resources could be targeted at recovering lost or under-represented genetic lines; these could then contribute to the future fitness of the population. Approaches of this type would be preferable to the indiscriminate generation of large numbers of identical individuals. Applying cloning technology to non-mammalian vertebrates may be more practical than attempting to use conventional reproductive technologies. As the scientific background to cloning technology was pioneered using amphibians, it may be possible to breed imminently threatened amphibians, or even restore extinct amphibian species, by the use of cloning. In this respect species with external embryonic development may have an advantage over mammals as developmental abnormalities associated with inappropriate embryonic reprogramming would not be relevant.
2003
The ability to produce cloned livestock using postmortem tissue could incorporate an additional application into the field of nuclear transfer. This study examined the feasibility of producing cloned cattle using a primary cell line established from a postmortem beef carcass. A market beef heifer processed at a USDA-certified slaughterhouse was used to develop a primary somatic cell line. Tissue samples were taken from the kidney and forelimb regions either 1) immediately following slaughter (fresh) or 2) 48 h postslaughter (cooled) where the carcass was housed at 2 to 4 • C. Tissue was removed and placed on ice in PBS + 5.0% (v:v) penicillin/streptomycin. A primary culture was established using standard techniques and cultured in supplemented DMEM F-12 medium. Once established, cells were trypsinized and either frozen or continually passaged. Cells used for nuclear transfer (NT) were passaged (48 h before use) and cultured with 15 µM roscovitine roughly 24 h prior to nuclear transfer. Cells were approximately 80% confluent and between passage numbers 1 and 11 at the time of NT. Selected slaughterhouse-derived oocytes were matured in supplemented TCM 199 medium for 18-20 h at 39 • C in 5.0% CO 2 and air. Mature Metaphase II oocytes were vortexed and stained with Hoechst 33342 to help with chromatin removal. Following enucleation, roscovitine-treated carcass cells were placed in the perivitelline space of the oocyte. Reconstructed NT embryos were fused in Zimmermann's medium and pulsed using needle-like electrodes. This was followed by activation using a combination of calcium ionophore (5 µM), cytochalasin D (5 µg mL −1), and cycloheximide (10 µg mL −1) in TCM + 10% FBS. Fused NT embryos were cultured in 50-µL drops of BARC medium (USDA, Beltsville, MD) for 7 days at 39 • C in a 5% CO 2 , 5% O 2 and 90% N 2 environment. Embryo development for all four groups (Table 1) was assessed with blastocysts (grade 1 or 2) being transferred into recipient cows 7 days post-estrus. Cleavage rates were not significantly different between groups, and the use of either fresh or cooled cells did not impact blastocyst formation. However, there was a significant difference (P = 0.05) in % blastocyst based on the source of the donor cell. Overall, one live calf resulted from 34 transferred NTs produced using kidney cells taken from a 48 h cooled carcass. These results display the feasibility of producing cloned calves from cells collected post mortem, which ultimately could be used as a tool to select breeding bulls based on their own steer carcass characteristics.
REPRODUCTION REVIEW Wildlife conservation and reproductive cloning Introduction
Reproductive cloning, or the production of offspring by nuclear transfer, is often regarded as having potential for conserving endangered species of wildlife. Currently, however, low success rates for reproductive cloning limit the practical application of this technique to experimental use and proof of principle investigations. In this review, we consider how cloning may contribute to wildlife conservation strategies. The cloning of endangered mammals presents practical problems, many of which stem from the paucity of knowledge about their basic reproductive biology. However, situations may arise where resources could be targeted at recovering lost or under-represented genetic lines; these could then contribute to the future fitness of the population. Approaches of this type would be preferable to the indiscriminate generation of large numbers of identical individuals. Applying cloning technology to non-mammalian vertebrates may be more practical than attempting to use conventional reproductive technologies. As the scientific background to cloning technology was pioneered using amphibians, it may be possible to breed imminently threatened amphibians, or even restore extinct amphibian species, by the use of cloning. In this respect species with external embryonic development may have an advantage over mammals as developmental abnormalities associated with inappropriate embryonic reprogramming would not be relevant.
Cloning and Stem Cells, 2009
Recent accomplishments in the fields of nuclear transfer and genomics, such as the cloned offspring production from frozen mouse cells, cryopreserved at not too low temperatures without cryoprotectors; or the sequencing of wooly mammoth genome, have opened the opportunity for the revival of extinct species. As expected, they are receiving a lot of publicity in the media and also scientific attention. Furthermore, it was recently published the ''revival'' of the first extinct subspecie: the Pyrenean ibex (Capra pyrenaica pyrenaica), a wild goat extinct in 2000. This strengthens the field of cloning as it had been tarnished by induced pluripotent stem cells (iPS) and other methods of reprogramming. However, for biological conservation purposes, cloning is not generally accepted as an alternative for animal conservation, and there is an ongoing debate between reproductive scientists and conservation specialists. Although we believe that nuclear transfer technologies have an opportunity in conservation efforts for some species that are on the brink of extinction and that population status, geographical isolation, reproductive characteristics, and human pressure create a situation that is almost unsustainable. In this article we discuss the barriers in cloning mammoths and cloning controversies in conservation from a zoological perspective, citing the species that might benefit from nuclear transfer techniques in the arduous journey so as not to disappear forever from this, our world.
Theriogenology, 2005
The objective of this study was to explore the feasibility of employing adult fibroblasts as donor cells in interspecies nuclear transfer (NT) between buffaloes and cattle. Buffalo and bovine oocytes matured in vitro for 22 h were enucleated by micromanipulation using the Spindle View system. An ear fibroblast, pretreated with 0.1 mg/mL aphidicolin for 24 h, followed by culture for 2-9 days in Dulbecco's Modified Eagle's Media + 0.5% fetal bovine serum, was introduced into the cytoplast by microinjection. Reconstructed oocytes were activated by exposure to 5 mM ionomycin for 5 min and 2 mM 6-dimethylaminopurine for 3 h. When buffalo adult fibroblasts were used as donor cells, there were no differences (P < 0.75) in the cleavage rate (66.2% versus 64.0%) between bovine and buffalo recipient oocytes, but more embryos derived from bovine cytoplasts developed to blastocysts than from buffalo cytoplasts (13.3% versus 3.0%, P < 0.05). When bovine adult fibroblasts were used as donor nuclei, both cleavage rate (45.3%) and blastocyst yield (4.5%) of NT embryos derived from buffalo cytoplasts were lower than those of NT embryos derived from bovine cytoplasts (65.5 and 11.9%, P < 0.05). The proportion of parthenogenetic buffalo (29.1%) or bovine (35.6%) oocytes developing to blastocysts was higher than those of NT embryos (P < 0.01). Interspecies NT embryos were derived from the donor cells and 55.0-61.9% of them possessed a normal diploid karyotype. In conclusion, embryos reconstructed by interspecies NT of adult fibroblasts between buffaloes and www.journals.elsevierhealth.com/periodicals/the
Mitochondrion, 2012
The fate of foreign mitochondrial DNA (mtDNA) following somatic cell nuclear transfer (SCNT) is still controversial. In this study, we examined the transmission of the heteroplasmic mtDNA of gaur donor cells and recipient bovine oocytes to an offspring and aborted and mummified fetuses at various levels during the development of gaur-bovine interspecies SCNT (iSCNT) embryos. High levels of the donor cell mtDNA were found in various tissue samples but they did not have any beneficial effect to the survival of iSCNT offspring. However, the factors on mtDNA inheritance are unique for each iSCNT experiment and depend on the recipient oocyte and donor cell used, which might play an important role in the efficiency of iSCNT.
2002
Bovine oocyte cytoplasm has been shown to support the development of nuclei from other species up to the blastocyst stage. Somatic cell nuclei from buffalo fetal ®broblasts have been successfully reprogrammed after transfer to enucleated bovine oocytes, resulting in the production of cloned buffalo blastocysts. The aim of this study was to compare the in vitro development of fetal and adult buffalo cloned embryos after the fusion of a buffalo fetal ®broblast, cumulus or oviductal cell with bovine oocyte cytoplasm. The fusion of oviductal cells with enucleated bovine oocytes was higher than that of fetal ®broblasts or cumulus cells (83% versus 77 or 73%, respectively). There was a signi®cantly higher cleavage rate (P < 0:05) for fused nuclear transferred embryos produced by fetal ®broblasts and oviductal cells than for cumulus cells (84 or 78% versus 68%, respectively). Blastocyst development in the nuclear transferred embryos produced by fetal ®broblasts was higher (P < 0:05) than those produced either by cumulus or oviductal cells. Chromosome analysis of cloned blastocysts con®rmed the embryo was derived from buffalo donor nuclei. This study demonstrates that nuclei from buffalo fetal cells could be successfully reprogrammed to develop to the blastocyst stage at a rate higher than nuclei from adult cells. #